The cloning strategies we have examined so far used HindIII for cloning, and thus the poly-His site was lost. As you remember, HindIII is NOT included within the original MCS of pQE-60. It was our decision to "add" it to the MCS sequence specified for the loneIt software (see illustration below).
If you go back to the CloneIt software and change the sequence of the cloning box from 113-157 (NcoI-HindIII) to  113-132 (NcoI-BglII), you may receive solutions resulting in His-tagged proteins. Try this later.

We'll now imply another strategy:  we'll use the Klenow DNA polymerase.

Read a short description of this enzyme and its use in cloning in the follwing website.
  

Go back to the "CloneIt" window. Leave eveything unchanged except the "parameters" section. Unmark "Just use classical enzymes", and mark "Use T4 DNA Pol.or Klenow".

Ten solutions are found.We will focus on solution 6/10.

Digest vector pQE-60 with BamHI [G^GATCC] (121) and BglII [A^GATCT] (127).

3'--G.GGA.G/GA.TC C.A/GA.TC T.CAT.CAC.CAT.CA--3'
5'--C.CCT.C CT.AG/G.T CT.AG/A.GTA.GTG.GTA.GT--5'

First digest with BglII. Then treat with Klenow DNA polymerase. Finally digest with BamHI.

Why do you have to follow this order of operation?

Resulting fragments following this pQE-60 digestion:

 1. 3425  bp BglII 127 - BamHI 121
 2.    6  bp BamHI 121 - BglII 127
 

Digest insert pGEM-GOI with BamHI [G^GATCC] (2915) and SmaI [CCC^GGG] (3284).

3'--C.TCC.G/GA.TC C.ATG.GTT.CTT.CC-.--C.GAA.CCC./GGG.GTA.CCG.AAT.TC--3'
5'--G.AGG.C CT.AG/G.TAC.CAA.GAA.GG-.--G.CTT.GGG./CCC.CAT.GGC.TTA.AG--5'

Is it necessary to treat pGEM-GOI with Klenow DNA polymerase after cutting it with SmaI ?

Resulting fragments following this pGEM-GOI digestion:

 1. 2973  bp SmaI 3284 - BamHI 2915
 2.  369  bp BamHI 2915 - SmaI 3284

Homework(Submit next week)

1. Fill in the table using the following list:
HindIII BglI,EcoRI,elements of the LacZ promotor,Poly-His,BglII,BglIII, EcoRI, NcoI, XhoI.
 

INCLUDED in pQE-GOI
EXCLUDED fromp pQE-GOI
   
   
   

2. Why can’t we use BglII both in pQE-60 and pGEM-GOI as a cloning procedure?

  1. This restriction site doesn’t exist in pGEM-GOI.
  2. BglII cuts more than once in pGEM-GOI.
  3. This restriction enzyme cuts GOI.
  4. BglII and BamHI share the common GATC site, which excludes this option.
Continue